1. Field of the Invention
The present invention relates generally to the field of cellular biochemistry and also to the field of diabetes. More particularly, it provides compositions and methods for inhibiting hexokinases in mammalian cells. Specifically provided are agents that stimulate the production of trehalose-6-phosphate; hexokinase-specific ribozymes and agents that competitively reduce hexokinase activity, e.g., by displacing hexokinase from mitochondria. Cells incorporating such agents and their respective genes, and advantageous methods of making and using cells with reduced hexokinase activity are also provided.
2. Description of the Related Art
The participation of the pancreatic islets of Langerhans in fuel homeostasis is mediated in large part by their ability to respond to changes in circulating levels of key metabolic fuels by secreting peptide hormones. Insulin secretion from islet .beta.-cells is stimulated by amino acids, three-carbon sugars such as glyceraldehyde and, most prominently, by glucose. The capacity of normal islet .beta.-cells to sense a rise in blood glucose concentration and to respond to elevated levels of glucose (as occurs following ingestion of a carbohydrate containing meal) by secreting insulin is critical to control of blood glucose levels. Increased insulin secretion in response to a glucose load prevents chronic hyperglycemia in normal individuals by stimulating glucose uptake into peripheral tissues, particularly muscle and adipose tissue.
Individuals in which islet .beta.-cell function is impaired suffer from diabetes. Insulin-dependent diabetes mellitus (IDDM, also known as Juvenile-onset, or Type I diabetes) represents approximately 15% of all human diabetes. IDDM is distinct from non-insulin dependent diabetes (NIDDM) in that only IDDM involves specific destruction of the insulin producing .beta.-cells of the islets of Langerhans in the pancreas. The destruction of .beta.-cells in IDDM appears to be a result of specific autoimmune attack, in which the patient's own immune system recognizes and destroys the .beta.-cells, but not the surrounding .alpha. (glucagon producing) or .delta. (somatostatin producing) cells that comprise the islet.
The precise events involved in .beta.-cell recognition and destruction in IDDM are currently unknown, but involve both the cellular and humoral components of the immune system. In IDDM, islet .beta.-cell destruction is ultimately the result of cellular mechanisms, in which cytotoxic T cells destroy .beta. cells which are erroneously perceived as foreign or harmful. The humoral component of the immune system, comprised of the antibody-producing B cells, is also inappropriately active in IDDM patients, who have serum antibodies against various .beta. cell proteins.
Glucose stimulates de novo insulin biosynthesis in .beta.-cells by increasing transcription, mRNA stability, translation, and protein processing. Glucose also rapidly stimulates the release of pre-stored insulin. Glucose transport into the .beta.-cell and metabolism of this sugar are absolute requirements for insulin secretion, leading to the hypothesis that its specific stimulatory effect is mediated by, and proportional to, its flux rate through glycolysis and related pathways.
The facilitated-diffusion type glucose transporter, GLUT-2, and the glucose phosphorylating enzyme, glucokinase, are known to be involved in the control of glucose metabolism in islet .beta.-cells (U.S. Pat. No. 5,427,940). Both proteins are members of gene families; GLUT-2 is unique among the five-member family of glucose transporter proteins in that it has a distinctly higher K.sub.m and Vmax for glucose. Glucokinase is the high K.sub.m and high Vmax counterpart of GLUT-2 among the family of hexokinases. Importantly, both proteins have affinities for glucose that allow dramatic changes in their activities over the physiological range of glucose. These proteins thus work in concert as the "glucose-sensing apparatus" that modulates insulin secretion in response to changes in circulating glucose concentrations by regulating glycolytic flux.
Treatment for IDDM is still centered around self-injection of insulin once or twice daily. The development of new therapeutic strategies is therefore necessary. The possibility of islet or pancreas fragment transplantation has been investigated as a means for permanent insulin replacement (Lacy et al., 1986). However, this approach has been severely hampered by the difficulties associated with obtaining tissue, as well as the finding that transplanted islets are recognized and destroyed by the same autoimmune mechanism responsible for destruction of the patients original islet .beta. cells.
U.S. Pat. No. 5,427,940 provided, for the first time, recombinant cells that secrete insulin in response to glucose. The generation of such artificial .beta. cells is achieved through the introduction of one or more genes selected from the insulin gene, the glucokinase gene and the GLUT-2 glucose transporter gene, so as to provide an engineered cell having all three of these genes in a biologically functional and responsive configuration.
The availability of the engineered cells of U.S. Pat. No. 5,427,940 makes cell-based insulin replacement therapy for IDDM a realistic goal. However, while evidently of significant use, it appears that these cells are not optimal for IDDM treatment owing to the fact that the glucokinase:hexokinase activity ratio in such cells is not likely to result in insulin secretion at physiological glucose concentrations. Accordingly, it is evident that improvements are needed in the engineering of cells for use in the treatment of diabetes and in other applications.